Nancy A. Haas

A novel MAP kinase regulates flagellar length in Chlamydomonas.

Little is known about the molecular basis of organelle size control in eukaryotes. Cells of the biflagellate alga Chlamydomonas reinhardtii actively maintain their flagella at a precise length. Chlamydomonas mutants that lose control of flagellar length have been isolated and used to demonstrate that a dynamic process keeps flagella at an appropriate length. To date, none of the proteins required for flagellar length control have been identified in any eukaryotic organism. Here, we show that a novel MAP kinase is crucial to enforcing wild-type flagellar length in C. reinhardtii. Null mutants of LF4 [2], a gene encoding a protein with extensive amino acid sequence identity to a mammalian MAP kinase of unknown function, MOK [3], are unable to regulate the length of their flagella. The LF4 protein (LF4p) is localized to the flagella, and in vitro enzyme assays confirm that the protein is a MAP kinase. The long-flagella phenotype of lf4 cells is rescued by transformation with the cloned LF4 gene. The demonstration that a novel MAP kinase helps enforce flagellar length control indicates that a previously unidentified signal transduction pathway controls organelle size in C. reinhardtii.

Molecular map of the Chlamydomonas reinhardtii nuclear genome.

ABSTRACT We have prepared a molecular map of the Chlamydomonas reinhardtii genome anchored to the genetic map. The map consists of 264 markers, including sequence-tagged sites (STS), scored by use of PCR and agarose gel electrophoresis, and restriction fragment length polymorphism markers, scored by use of Southern blot hybridization. All molecular markers tested map to one of the 17 known linkage groups of C. reinhardtii. The map covers approximately 1,000 centimorgans (cM). Any position on the C. reinhardtii genetic map is, on average, within 2 cM of a mapped molecular marker. This molecular map, in combination with the ongoing mapping of bacterial artificial chromosome (BAC) clones and the forthcoming sequence of the C. reinhardtii nuclear genome, should greatly facilitate isolation of genes of interest by using positional cloning methods. In addition, the presence of easily assayed STS markers on each arm of each linkage group should be very useful in mapping new mutations in preparation for positional cloning.

α-Tubulin gene family of maize (Zea mays L.) : evidence for two ancient α-tubulin genes in plants

Abstract Among 81 α-tubulin cDNA clones prepared from RNA from maize seedling shoot, endosperm and pollen, we identified six different α-tubulin coding sequences. The DNA sequence analysis of coding and non-coding regions from the clones showed that they can be classified into three different α-tubulin gene subfamilies. Genes within each subfamily encode proteins that are 99 to 100% identical in amino acid sequence. Deduced amino acid sequence identity between genes in different subfamilies ranges from 89 to 93 %. The results of hybridizations of genomic DNAs to α-tubulin coding region probes and to 3′ non-coding region probes constructed from six different α-tubulin cDNA clones indicated that the maize α-tubulin gene family contains at least eight members. Comparison of deduced α-tubulin amino acid sequences from maize and the dicot species Arabidopsis thaliana showed that α-tubulin isotypes encoded by genes in maize subfamilies I and II are more similar to specific Arabidopsis gene products (96 to 97% amino acid identity) than to isotypes encoded by genes in the other maize subfamilies. Phylogenetic analyses revealed that genes in these two subfamilies were derived from two ancient α-tubulin genes that predate the divergence of monocots and dicots. These same analyses revealed that the gene in maize subfamily III is more closely related to sequences from subfamily I genes than to those from subfamily IT genes. However, the subfamily III gene has no close counterpart in Arabidopsis. We found evidence of a transposable element-like insertion in the subfamily III gene in some maize lines.

The β-tubulin gene family in Zea mays: two differentially expressed β-tubulin genes

Maize β-tubulin are encoded by a large multigene family with at least nine members, as determined by Southern blot analysis. Two expressed genes, represented by the β1 genomic clone and the β2 cDNA clone, were examined in this study. The two genes encode β-tubulins which show 94% sequence identity at the amino acid level. Maize β1 transcript levels were highest in seedling root tip and tissue culture cells, which are both rapidly dividing tissues. No transcripts were detected in non-dividing leaf tissue. In contrast, β2 transcripts were present at relatively high levels in tissue culture cells and at lower levels in seedling root tip and leaf tissue. The electrophoretic mobility of the β2 polypeptide was examined in relation to the constellation of β-tubulin polypeptides on two-dimensional gel western blots of a maize pollen total protein extract. No evidence for post-translational modification of the β-tubulin polypeptides was found in pollen.

Characterization of four new β-tubulin genes and their expression during male flower development in maize (Zea mays L.)

Four different β-tubulin coding sequences were isolated from a cDNA library prepared from RNA from maize seedling shoots. The four genes (designated tub4, tub6, tub7 and tub8) represented by these cDNA clones together with the tub1 and tub2 genes reported previously encode six β-tubulin isotypes with 90–97.5% amino acid sequence identity. Results from phylogenetic analysis of 17 β-tubulin genes from monocot and dicot plant species indicated that multiple extant lines of β-tubulin genes diverged from a single precursor after the appearance of the two major subfamilies of α-tubulin genes described previously. Hybridization probes from the 3′ non-coding regions of six β-tubulin clones were used to quantify the levels of corresponding tubulin transcripts in different maize tissues including developing anthers and pollen. The results from these dot blot hybridization experiments showed that all of the β-tubulin genes were expressed in most tissues examined, although each gene showed a unique pattern of differential transcript accumulation. The tub1 gene showed a high level of transcript accumulation in meristematic tissues and almost no accumulation in the late stages of anther development and in pollen. In contrast, the level of tub4 transcripts was very low during early stages of male flower development but increased markedly (more than 100 times) during the development of anthers and in pollen. Results from RNAse protection assays showed that this increased hybridization signal resulted from expression of transcripts from one or two genes closely related to tub4. The tub4-related transcripts were not present in shoot tissue. Transcripts from the tub2 gene accumulated to very low levels in all tissues examined, but reached the highest levels in young anthers containing microspore mother cells. RNAse protection assays were used to measure the absolute levels of α- and β-tubulin transcripts in seedling shoot and in pollen. The α-tubulin gene subfamily I genes (tua1, tua2, tua4) contributed the great majority of α-tubulin transcripts in both shoot and pollen. Transcripts from the β-tubulin genes tub4, tub6, tub7, and tub8 were predominant in shoot, but were much less significant than the tub4-related transcripts in pollen.

The Hsp70 and Hsp40 Chaperones Influence Microtubule Stability in Chlamydomonas

Mutations at the APM1 and APM2 loci in the green alga Chlamydomonas reinhardtii confer resistance to phosphorothioamidate and dinitroaniline herbicides. Genetic interactions between apm1 and apm2 mutations suggest an interaction between the gene products. We identified the APM1 and APM2 genes using a map-based cloning strategy. Genomic DNA fragments containing only the DNJ1 gene encoding a type I Hsp40 protein rescue apm1 mutant phenotypes, conferring sensitivity to the herbicides and rescuing a temperature-sensitive growth defect. Lesions at five apm1 alleles include missense mutations and nucleotide insertions and deletions that result in altered proteins or very low levels of gene expression. The HSP70A gene, encoding a cytosolic Hsp70 protein known to interact with Hsp40 proteins, maps near the APM2 locus. Missense mutations found in three apm2 alleles predict altered Hsp70 proteins. Genomic fragments containing the HSP70A gene rescue apm2 mutant phenotypes. The results suggest that a client of the Hsp70–Hsp40 chaperone complex may function to increase microtubule dynamics in Chlamydomonas cells. Failure of the chaperone system to recognize or fold the client protein(s) results in increased microtubule stability and resistance to the microtubule-destabilizing effect of the herbicides. The lack of redundancy of genes encoding cytosolic Hsp70 and Hsp40 type I proteins in Chlamydomonas makes it a uniquely valuable system for genetic analysis of the function of the Hsp70 chaperone complex.